The interaction between collagen and platelets is the first event of the normal hemostatic response to injury. Collagen is the major extracellular matrix protein present in the subendothelium of blood vessels. Upon damage to the endothelium lining, as a consequence of injury to the vessel wall, collagen fibers, fibrous collagen I and III are exposed to platelets. This interaction leads to platelet adhesion, activation with a second phase of adhesion, secretion occurrence, and ultimately aggregation and development of a hemostatic plug (Kehrel et al., 1998, Blood 91:491-9).
The mechanism of collagen-platelet interactions is complex. It involves, on one hand, direct binding of collagen to specific platelet receptors (e.g., α2β1 integrin, collagen receptor, glycoprotein IV, and glycoprotein VI) and, on the other hand, indirect binding of collagen via bridging proteins (e.g., von Willebrand Factor (vWF)) that bind to both collagen and membrane receptors on platelets. Recent reports support a two-step mechanism of collagen-platelet interaction, consisting of platelet adhesion followed by platelet activation (Verkleij et al., 1998, Blood 91:3808-16). The first step involves the binding of collagen-bound vWF by the platelet receptor complex glycoprotein Ib/IX/V, followed by the direct binding of integrin α2β1 to collagen (Moroi et al., 1997, Thrombosis and Haemostasis 78:439-444 and Barnes et al., 1998, Current Opinion in Hematology 6:314-320). This step results in platelets adhering to the subendothelium of blood vessels under physiological conditions. The second step of collagen-platelet interaction involves another platelet collagen receptor, glycoprotein VI (Barnes et al., 1998, Current Opinion in Hematology 6:314-320). This binding leads to strengthening of attachment and platelet activation. It is believed that glycoprotein VI (GPVI) has a minor importance in the first step of adhesion but plays a major role in the second step of collagen-platelet interaction resulting in full platelet activation and consequently the formation of the platelet aggregates (Arai et al., 1995, British J. of Haematology 89:124-130).
Glycoprotein VI
Glycoprotein VI (GPVI) is a platelet membrane glycoprotein that is involved in platelet-collagen interactions. In particular, GPVI is a transmembrane collagen receptor expressed on the surface of platelets. GPVI has an apparent molecular mass of 58 kDa in its non-reduced form and 62 kDa after disulfide bond reduction as determined by its migration via SDS-PAGE. Treatment of platelets with N-glycanase has been shown to result in a faster migration of GPVI in SDS-PAGE by two kDa, which probably corresponds to only one N-glycosylation site.
The existence of a 62 kDa protein, later identified as GPVI, was first detected as an antigen recognized by the sera of a patient with steroid-responsive immune thrombocytopenic purpura associated with defective collagen-induced platelet functions (Sugiyama et al., 1987, Blood 69: 1712-1720). The patient's plasma, as well as a preparation of full length IgG antibodies, induced irreversible aggregation and ATP release in normal platelet-rich plasma. However, Fab fragments prepared from the serum of this patient blocked platelet aggregation induced by collagen (Sugiyama et al., 1987, Blood 69: 1712-172).
The importance of GPVI in platelet/collagen interactions was further confirmed by comparing the expression of platelet collagen receptors from a different patient, with a mild bleeding disorder, to that of a normal individual (Moroi et al., 1989, J. Clin. Invest. 84(5):1440-5). The patient's platelets lacked collagen-induced aggregation and adhesion, but retained normal aggregation and release by other agonists. The expression of a 61 kDa membrane glycoprotein was detected on non-reduced, two-dimensional SDS-PAGE, but was reduced compared to the expression levels found in a normal individual. This glycoprotein was termed glycoprotein VI (GPVI). The patient's platelets did not bind to types I and III collagen fibrils suggesting that GPVI functions as a collagen receptor involved in collagen-induced platelet activation and aggregation.
GPVI has been shown to be constitutively associated with the Fc receptor gamma (FcRγ), and FcRγ expression is lacking in GPVI-deficient platelets, suggesting that GPVI and FcRγ are co-expressed in platelets (Tsuji et al., 1997, J. Biol. Chem. 272:23528-31). Further, cross-linking of GPVI by F(ab′)2 fragments of anti-GPVI IgG has been shown to result in the tyrosine phosphorylation of the FcRγ-chain. FcRγ is tyrosine-phosphorylated upon platelet activation by collagen, collagen related peptide (CRP; Gibbins et al., 1997, FEBS Lett. 413:255-259) or the snake venom component convulxin that acts as a platelet agonist (Cvx; Lagrue et al., 1999, FEBS Letts. 448:95-100). Phosphorylation occurs on the immunoreceptor tyrosine-based activation motifs (ITAM) of FcRγ by kinases of the Src family (p59Fyn and p53/56 lyn) (Briddon S J and Watson, 1999, Biochem J. 338:203-9). Phosphorylation of FcRγ allows Syk, a signaling molecule, to bind and to be in turn phosphorylated and to activate phospholipase Cγ2 (PLCγ2). Further, platelet stimulation by collagen or Cvx have been shown to involve the association of phosphatidylinositol 3-kinase (PI3 kinase) and the adapter protein linker for activator of T cells (LAT) to the FcRγ (Carlsson et al., 1998, Blood 92:1526-31). Thus, FcRγ appears to interact with GPVI to effect signaling.
The results from the GPVI signal transduction pathway activation studies performed suggest that strong similarities exist between the GPVI signaling pathway in platelets and the one used by receptors for immune complexes, such as the high-affinity and low affinity receptors for IgG (FcRγI and FcRγIII), the high-affinity receptor for IgE (FcRεI) and the receptor for IgA (FcRαI) (Maliszewski et al., 1990, J. Exp. Med. 172:1665-72). These receptors also signal via the FcRγ chain and Syk. Expression of the FcRγI, FcRγIII has not been reported in platelets. The FcRγIIa seems to be the only IgG Fc-receptor consistently expressed on platelets, and it contains one ITAM. This receptor has been suggested to be involved in thrombocytopenia and thromboembolic complications of heparin-induced thrombocytopenia (HIT), the most common drug-induced immune thrombocytopenia (Carlsson et al., 1998, Blood 92:1526-31) and may also be involved in other immune thrombocytopenia such as immune thrombocytopenia purpura (Loscalzo, J., and Schafer, A. I., 1998, Thrombosis and Hemorrhage, J. Loscalzo and A. I. Schafer, eds., Baltimore: Williams and Wilkins).
Since its detection, the function of GPVI in platelet-collagen interactions and the signal transduction pathway induced by GPVI has been studied. However, the molecular cloning of GPVI has been elusive due, at least in part, to its extensive O-linked glycosylation. The inability to clone GPVI has limited the experiments that can be performed to better understand the role of GPVI in collagen-induced platelet activation and aggregation. Further, the development of treatments for disorders, such as bleeding disorders, resulting from mutations in GPVI or its promoter, have been hindered by the lack of knowledge about the nucleic acid and amino acid sequences of GPVI.